In Phlebotomus Sergenti (Diptera: Psychodidae)

726 The development of Psychodiella sergenti (Apicomplexa: Eugregarinorida) in Phlebotomus sergenti (Diptera: Psychodidae)

LUCIE LANTOVA1,2* and PETR VOLF1 1 Department of Parasitology, Faculty of Science and 2 Institute of Histology and Embryology, First Faculty of Medicine, Charles University in Prague, Czech Republic

(Received 1 October 2011; revised 17 November 2011; accepted 18 November 2011; first published online 8 February 2012)

SUMMARY

Psychodiella sergenti is a recently described specific pathogen of the sand fly Phlebotomus sergenti, the main vector of Leishmania tropica. The aim of this study was to examine the life cycle of Ps. sergenti in various developmental stages of the sand fly host. The microscopical methods used include scanning electron microscopy, transmission electron microscopy and light microscopy of native preparations and histological sections stained with periodic acid-Schiff reaction. Psychodiella sergenti oocysts were observed on the chorion of sand fly eggs. In 1st instar larvae, sporozoites were located in the ectoperitrophic space of the intestine. No intracellular stages were found. In 4th instar larvae, Ps. sergenti was mostly located in the ectoperitrophic space of the intestine of the larvae before defecation and in the intestinal lumen of the larvae after defecation. In adults, the parasite was recorded in the body cavity, where the sexual development was triggered by a bloodmeal intake. Psychodiella sergenti has several unique features. It develops sexually exclusively in sand fly females that took a bloodmeal, and its sporozoites bear a distinctive conoid (about 700 nm long), which is more than 4 times longer than conoids of the mosquito gregarines.

Key words: Psychodiella, Psychodiella sergenti, gregarine, Phlebotomus sergenti, sand ﬂy, life cycle, PAS, egg, larva, adult.

INTRODUCTION of the gregarines, the gamonts, can be found mostly in the larval gut lumen, where they undergo sexual Gregarines parasitizing sand flies (Apicomplexa: development; 2 gamonts associate into syzygy, which Eugregarinorida) are aseptate eugregarines recently is later enclosed in a cyst wall, forming a gametocyst. separated from the mosquito genus Ascogregarina Within the gametocyst, each gamont develops into Ward, Levine and Craig, 1982 to form a new genus gametes by gamogony, and after fertilization, during Psychodiella Votypka, Lantova and Volf, 2009 sporogony, zygotes differentiate into oocysts with 8 (Votypka et al. 2009). Despite their high degree sporozoites. In adults, the gregarines are located in of host specificity, only 5 Psychodiella species have the body cavity, undergoing sexual development. been described so far. Numerous studies on the In females, gametocysts attach to the accessory mosquito Ascogregarina species (e.g. Vavra, 1969; glands, and oocysts are injected into their lumen. Walsh and Callaway, 1969; Sanders and Poinar, During oviposition, the accessory gland fluid con- 1973; Munstermann and Levine, 1983; Chen et al. taining the oocysts adheres to the chorion of eggs and 1997a) showed that mosquito gregarines differ from serves as a source of infection for newly hatched sand fly gregarines at two critical points of the life larvae. This general life cycle is modified in other cycle: in mosquito larvae, the gregarines develop Psychodiella species, and differences were described intracellularly in the intestinal epithelial cells, and in in Psychodiella mackiei (Shortt and Swaminath, 1927) adults, they are located in the Malpighian tubules. and Psychodiella tobbi Lantova, Volf and Votypka, The life cycle of Psychodiella has been studied in 2010. detail in Psychodiella chagasi (Adler and Mayrink, Psychodiella sergenti Lantova, Volf and Votypka, 1961) by Adler and Mayrink (1961), Coelho and 2010 is a recently described specific pathogen of the Falcao (1964) and Warburg and Ostrovska (1991). sand fly Phlebotomus sergenti Parrot, 1917 (Diptera: Briefly, the 1st instar larvae are infected by swallow- Psychodidae), an important vector of Leishmania ing the gregarine infective stages, the oocysts. tropica (Wright, 1903) (e.g. Killick-Kendrick et al. Sporozoites released from oocysts reside in the larval 1995), which is a causative agent of human cutaneous midgut and develop into trophozoites. Mature stages leishmaniasis. Native preparations of sand fly adults revealed that sexual development of Ps. sergenti * Corresponding author: Lucie Lantova, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 (formation of syzygies, gametocysts and oocysts) Prague, Czech Republic. Tel: +420221951828. Fax: occurs only in female sand flies and is conditioned by +420224919704. E-mail: [email protected] a bloodmeal intake (Lantova et al. 2010). The aim of

Parasitology (2012), 139, 726–734. © Cambridge University Press 2012. The online version of this article is published within an Open Access environment subject to the conditions of the Creative Commons Attribution-NonCommercial-ShareAlike licence . The written permission of Cambridge University Press must be obtained for commercial re-use. Downloaded from doi:10.1017/S0031182011002411https://www.cambridge.org/core. IP address: 170.106.34.90, on 02 Oct 2021 at 18:12:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182011002411 Development of Psychodiella sergenti 727

the present study was to document the Ps. sergenti life in 2% osmium tetroxide in cacodylate buﬀer (4 °C), cycle in various life stages of its host, including the dehydrated through an ascending ethanol and eggs and 1st instar larvae. Several microscopical acetone series and embedded in Araldite 502/Poly- methods were used: scanning electron microscopy, Bed 812 (Polysciences Inc., Warrington, PA, USA). transmission electron microscopy and light micro- Thin sections (70 nm) were prepared on a Reichert- scopy of native preparations and histological sections Jung Ultracut E ultramicrotome (Leica Micro- stained with PAS reaction (periodic acid-Schiﬀ). systems GmbH, Wetzlar, Germany) and stained Using such a wide variety of microscopical tech- using uranyl acetate and lead citrate (Reynolds, niques provided a clear overview of the whole 1963). The sections were examined and photo- parasite’s life cycle. graphed using an electron microscope JEM-1011 (JEOL Ltd, Tokyo, Japan).

MATERIALS AND METHODS Histology Sand flies Larvae of 4th instar (BD and AD) and adults at The Phlebotomus sergenti colony infected with Ps. different days post-eclosion were dissected and fixed sergenti originated from females collected in Sanli at 4 °C in AFA fixative (96% ethanol: 38% formal- Urfa, Turkey. The colony maintenance was de- dehyde: acetic acid: distilled water; 12·5:1·5:1:10). scribed by Volf and Volfova (2011) and included an The head and the last posterior segments were egg-washing procedure using a series of solutions removed for better penetration of fixatives. (Poinar and Thomas, 1984) to reduce the infection Specimens were then washed 3 times in PBS and intensity of this pathogenic gregarine (Lantova et al. 70% ethanol and embedded in 2-hydroxyethyl 2011). The washing was omitted in a batch of eggs methacrylate (JB-4 Plus Embedding Kit, Poly- used for this study. sciences) following the manufacturer’s instructions. Histological sections (2–4 μm) were prepared using a Native preparations Shandon Finesse ME+microtome (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and stained Adults of both sexes and different ages were used, as using PAS (periodic acid-Schiff) reaction with well as 2 groups of 4th instar larvae: those with a gut Ehrlich’s acid haematoxylin: oxidization in 1% filled with larval diet are hereafter referred to as larvae periodic acid for 5 min, Schiff’s reagent (Sigma- before defecation (BD), and those ready to pupate Aldrich Corporation, St Louis, MO, USA) for with an empty gut are hereafter referred to as larvae 28 min, Ehrlich’s acid haematoxylin for 4 min (with after defecation (AD). The specimens were immobi- extensive washing in running water between steps). lized on ice and dissected in phosphate-buffered Stained sections were mounted on glass slides with saline (PBS) under a stereomicroscope SZX-12 Plastic UV Mount Mounting Media (Polysciences) (Olympus Corporation, Tokyo, Japan). Micro- and observed and photographed under an optical graphs were produced with a DP-70 digital camera microscope BX-51 (Olympus) connected to a DP-70 (Olympus) connected to an optical microscope BX- digital camera (Olympus). 51 (Olympus). RESULTS Scanning microscopy Ps. sergenti oocysts on sand fly eggs Gravid sand fly females were left to oviposit into a The exochorion of Ph. sergenti eggs was contaminated plastic cup filled with plaster (commonly used for the with Ps. sergenti oocysts (Fig. 1). The number of colony, see Volf and Volfova, 2011), and 1–2 days oocysts per egg varied; some eggs appeared without later, eggs were transferred using a fine brush onto a any contamination, others contained dozens of double-sided tape. Oocysts were documented on the oocysts. The distribution of oocysts on the chorion surface of Ph. sergenti eggs using a scanning was not even, they usually concentrated around the tip microscope TM-1000 (Hitachi High-Technologies of the egg (Fig. 1A – D) or along the longitudinal axis Corporation, Tokyo, Japan). This scanning micro- of the egg in contact with the exochorion sculpturing scope does not require any sample preparation and ridges (Fig. 1A – C). Manipulation of eggs by a brush allows direct observation of unfixed and non- occasionally caused detachment of the oocysts from desiccated samples. the egg surface. In some cases, distinct imprints of the detached oocysts were visible (Fig. 1E). Electron microscopy Ps. sergenti sporozoites in 1st instar sand fly larvae Three-day-old 1st instar larvae were fixed in modified Karnovsky fixative (Karnovsky, 1965) or in 2·5% In 1st instar larvae, Ps. sergenti sporozoites were glutaraldehyde in cacodylate buffer (4 °C), post-fixed found in the intestine (Fig. 2). They were located

Downloaded from https://www.cambridge.org/core. IP address: 170.106.34.90, on 02 Oct 2021 at 18:12:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182011002411 Lucie Lantova and Petr Volf 728

Fig. 1. Scanning electron micrographs of Phlebotomus sergenti eggs infected with Psychodiella sergenti.(A– D) Psychodiella sergenti oocysts (arrows) attached to the chorion of eggs. (E) Imprints of the detached oocysts. Scale bars (A)=50 μm, (B – D)=25 μm, (E)=10 μm.

mostly in the posterior midgut, exclusively in the Ps. sergenti development in sand fly males ectoperitrophic space between the peritrophic matrix Even though males up to 13 days post-eclosion were and the epithelium (Fig. 2A). The sporozoites examined, observations of both the native prep- appeared to be gliding on microvilli, or they were arations and histological sections recorded only attached to the epithelial cells with a distinct mucron gamonts (Fig. 4A – C). The gamonts were usually (Fig. 2B, E). They had a 3-layered pellicle (Fig. 2D), round or oval, but in high-intensity infections, some tightly arranged subpellicular microtubules (Fig. 2C) had a shape of an hour-glass or a tear-drop. They and posteriorly located nucleus (Fig. 2E). A very were found mostly in the body cavity, in several cases distinct conoid (approximately 700 nm long) as well also in the fat body but never in the intestine or as a polar ring and numerous micronemes were elsewhere. The characteristic appearance of the observed (Fig. 2E, F). The sporozoites were never gregarines with distinctive nucleus (Fig. 4B) allowed located intracellularly. them to be easily distinguished from rectal papilla, the PAS-positive tissue of adult sand flies (Fig. 4A). Ps. sergenti development in 4th instar sand fly larvae The documentation of Ps. sergenti in 4th instar larvae Ps. sergenti development in sand fly females was accomplished in native preparations (Fig. 3A, B) and histological sections stained with PAS reaction In females that did not take a bloodmeal, no other with Ehrlich’s acid haematoxylin (Fig. 3C, D). gregarine stages except gamonts were found, even The gregarine stages found in 4th instar larvae though the females were dissected up to the age of were mostly gamonts, occasionally also syzygies and 13 days. On the other hand, in blood-fed females, gametocysts but never oocysts. In BD larvae, the whole sexual development, including syzygies, gamonts were documented in the ectoperitrophic gametocysts and oocysts, was demonstrated space of the intestine, mainly in the posterior midgut (Figs 4D – F and 5). Gamonts and gametocysts (Fig. 3A, C), while in AD larvae the gregarines were were located in the body cavity and a few gamonts, located in the midgut lumen along its whole length particularly when the infection intensity was high, in (Fig. 3B, D). In a few cases, gamonts were found also the fat body. in the larval body cavity, but no intracellular In females 2 days post-bloodmeal, young gameto- development or cell damage was detected. cysts were found still consisting of the 2 original The PAS reaction proved to be very useful in gamonts with their nuclei (Fig. 5A). From 3 to 5 days highlighting gregarines in the host tissues. Their after a bloodmeal, gametocysts at different stages of PAS-positive amylopectin granulation was typical maturation were documented (Fig. 5D, E), some of and easily recognizable from other PAS-positive them being attached to the accessory glands (Figs 4D objects, e.g. from the midgut contents (Fig. 3C). and 5B – E). Around day 5, the gametocysts were

Fig. 2. Transmission electron micrographs of the intestine of 1st instar Phlebotomus sergenti larvae infected with Psychodiella sergenti. (A) Unattached sporozoite (arrow) in the ectoperitrophic space of the intestine. (B, E, F) Sporozoites attached to the epithelial cells of the intestine. (C) Transverse section of a sporozoite. (D) Detail of a 3-layered pellicle of a sporozoite. CO, conoid; IT, lumen of the larval intestine with organic debris and yeasts; MN, micronemes; MT, subpellicular microtubules; MU, mucron; NU, nucleus; PM, peritrophic matrix; PR, polar ring. Scale bars (A)=2 μm, (B, E)=1 μm, (C)=300 nm, (D)=100 nm, (F)=500 nm.

fully matured and, later, the accessory glands in which gametocysts are formed in adults of both of blood-fed females became filled with oocysts sexes, including females that did not take a bloodmeal (Figs 4E, F and 5F – H). (Coelho and Falcao, 1964; Lantova et al. 2010). Rectal papilla (Fig. 5A) and oocytes (Fig. 5D – F) The only other sand fly gregarine with the life cycle in are the strongly PAS-positive tissues found in adult hosts similar to Ps. sergenti is an undescribed females; however, gregarines stained with PAS parasite reported by Ayala (1971)inLutzomyia reaction were distinct, particularly in sections not vexatrix occidentis Fairchild and Hertig, 1957. post-stained with Ehrlich’s acid haematoxylin Similar to Ayala (1973), we hypothesize that the (Fig. 5B, C). hormonal changes influenced by the ingestion of blood trigger the sexual cycle of the gregarine. In Ps. chagasi, Coelho and Falcao (1964) and DISCUSSION Warburg and Ostrovska (1991) found gregarine The main distinctive feature of the Ps. sergenti life oocysts in 4th instar larvae, the former authors even cycle is the fact that the gregarines develop sexually described the formation of oocysts that were being exclusively in adult females that had a bloodmeal. defecated and served as a source of horizontal This is a striking contrast to Ps. chagasi and Ps. tobbi, infection to other larvae. Contrastingly, no oocysts

Fig. 3. Native preparations of the intestine (A, B) and histological sections of the whole body stained with PAS reaction with Ehrlich’s acid haematoxylin (C, D) of Phlebotomus sergenti 4th instar larvae infected with Psychodiella sergenti. (A, C) 4th instar larva before defecation. The gregarines (arrows) are located in the ectoperitrophic space of the intestine. (B, D) 4th instar larva after defecation. The gregarines (arrows) are located in the lumen of the intestine. FB, fat body; IT, intestine; MT, Malpighian tubules. Scale bars=100 μm.

were found in 4th instar larvae of Ps. tobbi (Lantova In adults, the main location of the gregarines et al. 2010)orPs. sergenti in this study. The was the body cavity, as also recorded by Shortt and localization of gregarines in the ectoperitrophic Swaminath (1927), Adler and Mayrink (1961) and space of 4th instar BD larvae was observed in this Ostrovska et al. (1990). In high-intensity infections, study and also by other authors (Shortt and we found Ps. sergenti also in the fat body. The Swaminath, 1927; Coelho and Falcao, 1964; attachment of the gametocysts to the accessory glands Warburg and Ostrovska, 1991). of females observed in Ps. sergenti was recorded in all

Fig. 4. Histological sections stained with PAS reaction with Ehrlich’s acid haematoxylin of Phlebotomus sergenti males infected with Psychodiella sergenti (A, B) and native preparations of Ph. sergenti adults infected with Ps. sergenti (C – F). (A) Male body cavity filled with gamonts (arrows). (B) Gamonts with distinctive nuclei in the male body cavity (arrows). (C) Gamonts from the body cavity of a male sand fly. (D) Gametocyst (arrow) attached to the accessory gland of a female 5 days post-bloodmeal. (E) Accessory gland of a female 8 days post-bloodmeal filled with oocysts. (F) Detail of oocysts. AG, accessory glands; IT, intestine; NG, neural ganglion; RP, rectal papilla; VS, vesicular seminalis. Scale bars (A, C – E)=100 μm, (B)=50 μm, (F)=10 μm.

other sand fly gregarine species (reviewed by recorded in the sand fly larvae. This is in agreement Ostrovska et al. 1990). with findings of various authors on Ps. chagasi, Psychodiella sergenti sporozoites in 1st instar but in contrast to the findings of Shortt and larvae were either in contact with the microvilli Swaminath (1927)onPs. mackiei, where intra- or attached to the epithelial cells with a mucron. cellular stages were reported in the gut of 1st instar No intracellular development of Ps. sergenti was larvae.

Fig. 5. Histological sections of Phlebotomus sergenti females 2 (A – C), 3 (D, E) and 7 (F – H) days post-bloodmeal infected with Psychodiella sergenti stained with PAS reaction with Ehrlich’s acid haematoxylin. (A) Young gametocyst in the body cavity of a female 2 days post-bloodmeal. (B) Gametocysts (arrows) attached to the accessory glands of a female 2 days post-bloodmeal, stained only with PAS reaction. (C) Section B post-stained with Ehrlich’s acid haematoxylin. (D) Gamonts and gametocysts (arrow) in the body cavity of a female 3 days post-bloodmeal. (E) Gametocysts (arrows) at diﬀerent stages of maturation attached to the accessory glands of a female 3 days post-bloodmeal. (F) Female 7 days post-bloodmeal with gamonts (arrow) in the body cavity and oocysts in the accessory glands. (G) Accessory gland ﬁlled with gregarine oocysts. (H) Detail of oocysts. AG, accessory glands; IT, intestine; OC, oocytes; RP, rectal papilla. Scale bars (A, G)=50 μm, (B – F)=100 μm, (H)=10 μm.

The conoid of Ps. sergenti sporozoite has a typical by being located in the ectoperitrophic space. This coiled appearance and is strikingly long (approxi- helps the parasite to sustain a certain level of infection mately 700 nm). This is in accordance with obser- during pupation. (3) The injection of the oocysts into vations performed on Ps. chagasi (Warburg and the accessory gland lumen, facilitated by the host Ostrovska, 1991), where the conoid was around immune response (Warburg and Ostrovska, 1989), is 700 nm long. On the other hand, shorter conoids a unique mode of vertical transmission. (4) The most were recorded in mosquito ascogregarines (approxi- remarkable feature is the fact that Ps. sergenti does not mately 150 nm – Sheffield et al. 1971; Chen et al. develop sexually in males or females without a 1997b) and some other apicomplexans (approximately bloodmeal, which is advantageous for the gregarines, 250 nm – e.g. Roberts et al. 1970; Dubey et al. 1998). as they only invest energy into the sexual develop- This suggests that longer conoids might be typical for ment where the vertical transmission is possible, i.e. sand fly gregarines. The organization of the subpelli- in blood-fed females. cular microtubules of Ps. sergenti resembles Ps. chagasi (Warburg and Ostrovska, 1991) and differs ACKNOWLEDGEMENTS from ascogregarines; both Psychodiella species have a high number of tightly arranged subpellicular micro- We would like to thank Associate Professor Radomira tubules, while Ascogregarina culicis Ross, 1989 pos- Vagnerova (First Faculty of Medicine, Charles University ffi in Prague, Czech Republic) for valuable advice on sesses 21 of them (She eld et al. 1971). The surface of histological methods, Professor Jaromir Plasek (Faculty of Ps. sergenti sporozoites consists of a 3-layered pellicle. Mathematics and Physics, Charles University in Prague, Previously, a 2-layered pellicle was reported by Czech Republic) for advice and the opportunity to use the Warburg and Ostrovska (1991)inPs. chagasi and by Hitachi TM-1000 scanning microscope and Professor ’ Sheffield et al. (1971) and Sanders and Poinar (1973) Joseph Schrevel (Museum National d Histoire Naturelle, Paris, France), Dr Andrea Valigurova (Faculty of Science, in mosquito gregarine species. As pointed out by Masaryk University, Brno, Czech Republic) and Professor Vavra (1969), such a difference might be due to the fact Jiri Vavra (Academy of Sciences of the Czech Republic, that the 2 inner membranes could sometimes be very Ceske Budejovice, Czech Republic) for advice on method- close, giving the impression of a single membrane. ology and for stimulating discussion. Scanning electron microscopy showed Ps. sergenti

oocysts frequently attached to the longitudinal exo- FINANCIAL SUPPORT chorion sculpturing ridges of the sand flyeggs.In contrast, Adler and Mayrink (1961) recorded Ps. This study was supported by the Czech Ministry of Education (projects MSM0021620828, and LC06009). chagasi oocysts adhered to the Lutzomyia longipalpis (Lutz and Neiva, 1912) egg surface at a right angle to the longitudinal axis. This suggests that, similar REFERENCES fi to the species-speci cchorionornamentation(e.g. Adler, S. and Mayrink, W. (1961). A gregarine, Monocystis chagasi n. sp., De Almeida et al. 2004), also the character of of Phlebotomus longipalpis. Remarks on the accessory glands of P. longipalpis. the oocyst contamination might be species specific. Revista do Instituto de Medicina Tropical de Sao Paulo 3, 230–238. Ayala, S. C. (1971). Gregarine infections in the California sandfly, The location of oocysts seems to be connected to the Lutzomyia vexatrix occidentis. Journal of Invertebrate Pathology 17, process of the exochorion formation during ovipos- 440–441. doi: 10.1016/0022-2011(71)90020-6 ition, when it is secreted by the accessory glands. The Ayala, S. C. (1973). The phlebotomine sandfly-protozoan parasite com- munity of central California grasslands. American Midland Naturalist 89, viscous consistency of the secretion enables the oocysts 266–280. doi: 10.2307/2424032 to adhere to the egg surface at the site where drying Chen, W. J., Chow, C. Y. and Wu, S. T. (1997a). Ultrastructure of exochorion produces characteristic sculpturing ridges. infection, development and gametocyst formation of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in its mosquito host, Aedes albopictus The Psychodiella life cycle has been studied in (Diptera: Culicidae). Journal of Eukaryotic Microbiology 44, 101–108. doi: detail by a limited number of authors, and only a 10.1111/j.1550-7408.1997.tb05945.x single report has been published documenting this Chen, W. J., Wu, S. T., Chow, C. Y. and Yang, C. H. (1997b). Sporogonic development of the gregarine Ascogregarina taiwanensis (Lien and Levine) parasite (Ps. chagasi) using electron microscopy (Apicomplexa: Lecudinidae) in its natural host, Aedes albopictus (Skuse) (Warburg and Ostrovska, 1991). In the present (Diptera: Culicidae). Journal of Eukaryotic Microbiology 44, 326–331. doi: study, we used various microscopical methods in 10.1111/j.1550-7408.1997.tb05674.x fl Coelho, M. de V. and Falcao, A. L. (1964). Aspects of the life-cycle of major sand y developmental stages giving a self- “Monocystis chagasi” Adler and Mayrink, 1961, in “Phlebotomus long- contained overview of the Ps. sergenti life cycle. There ipalpis”. Revista Brasileira de Biologia 24, 417–421. are several features suggesting a close relationship De Almeida, D. N., Oliveira, R. da S., Brazil, B. G. and Soares, M. J. fl (2004). Patterns of exochorion ornaments on eggs of seven South American between Ps. sergenti and its sand y host, supporting species of Lutzomyia sand flies (Diptera: Psychodidae). Journal of Medical the hypothesis about co-evolution of gregarines and Entomology 41, 819–825. doi: 10.1603/0022-2585-41.5.819 sand flies as mentioned by Ostrovska et al. (1990). (1) Dubey, J. P., Lindsay, D. S. and Speer, C. A. (1998). Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and The attachment of the oocysts to the exochorion is development of tissue cysts. Clinical Microbiology Reviews 11, 267–299. possibly closely related to the exochorion formation, Karnovsky, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high facilitating the vertical transmission of the parasite. osmolality for use in electron microscopy. Journal of Cell Biology 27, 137A–138A. (2) The gregarine is protected from the expulsion Killick-Kendrick, R., Killick-Kendrick, M. and Tang, Y. (1995). from the larval intestine during pre-pupal defecation Anthroponotic cutaneous leishmaniasis in Kabul, Afghanistan: the high

susceptibility of Phlebotomus sergenti to Leishmania tropica. Transactions of mosquito Aedes sierrensis (Ludlow). Journal of Protozoology 20, 594–602. the Royal Society of Tropical Medicine and Hygiene 89, 477. doi: 10.1016/ doi: 10.1111/j.1550-7408.1973.tb03582.x 0035-9203(95)90072-1 Sheffield, H. G., Garnham, P. C. C. and Shiroishi, T. (1971). The fine Lantova, L., Ghosh, K., Svobodova, M., Braig, H. R., Rowton, E., structure of the sporozoite of Lankesteria culicis. Journal of Protozoology 18, Weina, P., Volf, P. and Votypka, J. (2010). The life cycle and host 98–105. doi: 10.1111/j.1550-7408.1971.tb03289.x specificity of Psychodiella sergenti n. sp. and Ps. tobbi n. sp. (Protozoa: Shortt, H. E. and Swaminath, C. S. (1927). Monocystis mackiei n. sp. Apicomplexa) in sand flies Phlebotomus sergenti and Ph. tobbi (Diptera: parasitic in Phlebotomus argentipes, Ann. and Brun. Indian Journal of Psychodidae). Journal of Invertebrate Pathology 105, 182–189. doi: 10.1016/ Medical Research 15, 539–552. j.jip.2010.07.001 Vavra, J. (1969). Lankesteria barretti n. sp. (Eugregarinida, Diplocystidae), Lantova, L., Svobodova, M. and Volf, P. (2011). Effects of Psychodiella a parasite of the mosquito Aedes triseriatus (Say) and a review of the genus sergenti (Apicomplexa, Eugregarinorida) on its natural host Phlebotomus Lankesteria Mingazzini. Journal of Protozoology 16, 546–570. doi: 10.1111/ sergenti (Diptera, Psychodidae). Journal of Medical Entomology 48, 995–990. j.1550-7408.1969.tb02314.x doi: 10.1603/ME11018 Volf, P. and Volfova, V. (2011). Establishment and maintenance of sand fly Munstermann, L. E. and Levine, N. D. (1983). Ascogregarina geniculati colonies. Journal of Vector Ecology 36 (Suppl.) S1–S9. doi: 10.1111/j.1948- sp. n. (Protozoa, Apicomplexa) from the mosquito Aedes geniculatus. The 7134.2011.00106.x Journal of Parasitology 69, 769–772. doi: 10.2307/3281155 Votypka, J., Lantova, L., Ghosh, K., Braig, H. and Volf, P. (2009). Ostrovska, K., Warburg, A. and Montoya-Lerma, J. (1990). Molecular characterization of gregarines from sand flies (Diptera: Ascogregarina saraviae n. sp. (Apicomplexa: Lecudinidae) in Lutzomyia Psychodidae) and description of Psychodiella n. gen. (Apicomplexa: lichyi (Diptera: Psychodidae). Journal of Protozoology 37,69–70. doi: Gregarinida). Journal of Eukaryotic Microbiology 56, 583–588. doi: 10.1111/j.1550-7408.1990.tb05872.x 10.1111/j.1550-7408.2009.00438.x Poinar, G. O., Jr. and Thomas, G. M. (1984). Bacteria. In Laboratory Walsh, R. D., Jr. and Callaway, C. S. (1969). The fine structure of the Guide to Insect Pathogens and Parasites (ed. Thomas, G.M.), pp. 79–104. gregarine Lankesteria culicis parasitic in the yellow fever mosquito Aedes Plenum Press, New York, USA. aegypti. Journal of Protozoology 16, 536–545. doi: 10.1111/j.1550- Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron- 7408.1969.tb02313.x opaque stain in electron microscopy. Journal of Cell Biology 17, 208–212. Warburg, A. and Ostrovska, K. (1989). An immune response-dependent doi: 10.1083/jcb.17.1.208 mechanism for the vertical transmission of an entomopathogen. Experientia Roberts, W. L., Hammond, D. M. and Speer, C. A. (1970). 45, 770–772. doi: 10.1007/BF01974585 Ultrastructural study of the intra- and extracellular sporozoites of Eimeria Warburg, A. and Ostrovska, K. (1991). Host-parasite relation-ships of callospermophili. The Journal of Parasitology 56, 907–917. doi: 10.2307/ Ascogregarina chagasi (Eugregarinorida, Aseptatorina, Lecudi- 3277504 nidae) in Lutzomyia longipalpis (Diptera: Psychodidae). Inter- Sanders, R. D. and Poinar, G. O., Jr. (1973). Fine structure and life national Journal for Parasitology 21,91–98. doi: 10.1016/0020-7519(91) cycle of Lankesteria clarki sp. n. (Sporozoa: Eugregarinida) parasitic in the 90124-P